MIS5-1 - The Roles of Growth Arrest DNA Damage-Inducible Gene 45Γ (GADD45Γ) Expression in Sorafenib-Induced Apoptosis in Hepatocellular Carcinoma (HCC) Cells

Abstract

ABSTRACT Background We previously found that GADD45b, which is associated with cellular stress response and apoptosis regulation, contributes to sorafenib resistance in HCC cells. The present study explored the role of GADD45γ, another GADD45 family protein, in HCC treatment. Methods HCC cell lines tested included Huh-7, HepG2, Hep3B, Huh-7R, HepG2R, the latter two sorafenib-resistant cell lines established by continuously exposure of Huh-7 and HepG2 cells to sorafenib. GADD45γ expression was examined by quantitative real-time PCR (qRT-PCR) and western blotting. Control of GADD45γ transcription was examined by luciferase reporter assay using a series of reporter plasmids with deletions or site-directed mutants of the 5′-flanking region of GADD45γ promoter. Apoptosis was analyzed by flow cytometry and western blotting. Expression of GADD45γ in human HCC tumor tissues was measured by qRT-PCR and immunohistochemistry. Results GADD45γ mRNA and protein levels were increased after sorafenib independent of cellular MEK/ERK activity. GADD45γ induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6-7 µM) than in sorafenib-resistant HCC cells (Hep3B, Huh-7R and HepG2R, IC50 12-15 µM). Knockdown of GADD45γ expression partially abrogated the pro-apoptotic effects of sorafenib in sorafenib-sensitive cells but not sorafenib-resistant cells. The region -449/ - 82 in the 5′-flanking region of GADD45γ promoter was found crucial for GADD45 gamma induction by sorafenib. Decreased GADD45γ expression in HCC tumor tissue was associated with worse overall survival in patients who underwent surgery for HCC. Conclusions Induction of GADD45γ expression contributes to sorafenib-induced apoptosis in HCC cells. The role of GADD45γ as a predictive biomarker for sorafenib sensitivity in HCC warrants further exploration. (Supported by grants NSC-100-2325-B-002 -042, NSC 100-2314-B-002 -058-MY3, DOH100-TD-B-111-001, and NHRI-EX101-9911BC.)